ent-Kaurene, ent-kauran-16-ol, and shunted diterpenoids from an engineered strain of Aspergillus oryzae.

Aspergillus oryzae was engineered to produce high levels of geranylgeranyldiphosphate (GGPP) and ent-kaurene, via ent-copalyl diphosphate, as precursors for future metabolic engineering projects via the expression of ggs2 and cps/ks genes from Gibberella fujikuroi under the control of strong constitutive promoters. GGPP was shunted to three new metabolites that were fully identified by NMR and HRMS. ent-Copalyl diphosphate was shunted to a further seven new compounds that were also identified by NMR and HRMS. The titre of ent-kaurene was significantly increased by heterologous expression of a truncated hydroxymethylglutaryl CoA reductase gene from Avena strigosa (AstHMGR), while replacement of the dual terpene cyclase encoding gene cps/ks with the individual cyclase genes, ptmT2 from Streptomyces platensis and Bjks from Bradyrhizobium japonicum, resulted mainly in accumulation of ent-copalyl diphosphate-derived shunt products. Finally, ent-kauran-16-ol was biosynthesised by coexpression of ggs2 with the bacterial cyclases ptmT2 and ptmT3.